Methacrylic acid- containing beads modulate macrophage polarization in a vascularizing subcutaneous mouse model

摘要

Previously, polymeric beads containing methacrylic acid (MAA) have been used to accelerate vascularization in mice. However, the mechanism(s) of this highly beneficial phenomenon are unclear. Studies in a diabetic wound healing model in vivo showed augmented vessel formation and accelerated wound closure, despite no apparent increase in macrophage infiltration. Vascularization has been linked to macrophage polarization and function, suggesting that perhaps an advantageous shift in macrophage polarization may be the impetus for this favorable response. Given this, we hypothesized that MAA beads shift the dynamic sequence in macrophage polarization towards the vascular reparative phenotype to promote vessel formation. To test our hypothesis, we subcutaneously injected MAA beads and control methyl methacrylate (MAA) beads into CD1 mice for 1-7 days (using PEG avg. wt. 1450 as a vehicle). The beads and surrounding tissue were explanted and analyzed for the infiltration of inflammatory cells and the formation of vessels by flow cytometry and histology, respectively. MAA beads induced a polarization bias towards MHCII-CD206+ ("M2") macrophages (Fig. 1); which was accompanied by significantly more CD31+vessels (Fig. 2), compared to controls. To fully demonstrate that the newly formed MM-mediated vessels were perfusable and anastomosed with the host vasculature, we injected fluorophore-conjugated mouse GSL-1 lectin into mice 7 days after bead injection and visualized the mouse vasculature using confocal microscopy following CLARITY, a tissue preparation technique that enabled high-resolution imaging of intact tissues. Fluorophore staining was observed surrounding MM beads but not MM beads in CLARITY-processed tissues, suggesting that vessels were anastomosed to host vessels and perfusable (Fig. 2). These data support the hypothesis that macrophage polarization is a key element of MAA-mediated vessel formation. This information is expected to facilitate the development of advanced therapeutic biomaterials with vascular regenerative properties.